Listeria monocytogenes and Shigella flexneri are human intestinal pathogens that replicate in the cytosol of infected cells and spread directly from primarily infected cells to neighboring cells. The dissemination process is a fundamental aspect of pathogenesis and spreading-defective bacterial mutants are essentially avirulent. The ability of L. monocytogenes and S. flexneri to spread from cell to cell is related to their ability to dispay actin-based motility in the cytosol of infected cells. These bacteria produce virulence factors tha lead to the recruitment at their surface of an essential host cell actin nucleator, the ARP2/3 complex. This results in actin polymerization at one pole of the bacteria, which propels the rods throughout the cytosol. Seminal electron microscopy studies revealed that, when bacteria reach the cell cortex, they form plasma membrane extensions that protrude into the cytosol of neighboring cells. These protrusions are resolved in the receiving cells into double membrane vacuoles, from which the pathogens escape by producing virulence factors that disrupt the integrity of eukaryotic membranes. In contrast to our advanced understanding of the molecular mechanisms supporting cytosolic actin-based motility, the mechanisms supporting pathogen dissemination through membrane protrusion formation are unresolved. To address this gap in knowledge, we have developed innovative procedures for imaging intracellular pathogen dissemination and identified bacterial and cellular factors specifically required for the formation/resolution of membrane protrusions and double membrane vacuoles upon L. monocytogenes infection. Here, we propose to gain the first mechanistic insight into (Aim1) the cellular factors involved in actin network formation in L. monocytogenes protrusions, (Aim2) the bacterial factors involved in L. monocytogenes protrusion formation and resolution, (Aim3) the cellular factors involved in L. monocytogenes double membrane vacuole maturation and (Aim4) the bacterial and cellular factors supporting S. flexneri dissemination.